Compositions containing peptides, optionally conjugated to nucleic acid-binding groups, to lipids or to dendrimers, and transfection agents, including cationic lipids and dendrimer polymers, useful for transfecting eukaryotic cells are disclosed. Also disclosed are methods of transfecting eukaryotic cells employing such compositions.
Lipid aggregates such as liposomes can function to facilitate introduction of macromolecules, such as DNA, RNA, and proteins, into living cells. Lipid aggregates comprising cationic lipid components can be effective for delivery and introduction of large anionic molecules, such as nucleic acids, into certain types of cells. See Felgner, P. L. and Ringold, G. M. (1989) Nature 337:387-388 and Felgner, P. L. et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413. Since the membranes of most cells have a net negative charge, anionic molecules, particularly those of high molecular weight, are not readily taken up by cells. Cationic lipids aggregate to and bind polyanions, such as nucleic acids, tending to neutralize the negative charge. The effectiveness of cationic lipids in transfection of nucleic acids into cells is thought to result from an enhanced affinity of cationic lipid-nucleic acid aggregates for cells, as well as the function of the lipophilic components in membrane fusion.
Dendrimers are a new type of synthetic polymer with regular, dendric branching with radial symmetry composed of an initiator core, interior layers (or generations) of repeating units, radially attached to the core and an exterior surface of terminal functional groups. See: D. A. Tomalia and H. D. Durst (1993) in E. Weber (ed.) Topics in Current Chemistry, Vol. 165: Supramolecular Chemistry I-Directed Synthesis and Molecular Recognition, Springer-Verlag, Berlin, pp.193-313. The size, shape and surface charge density of the dendrimer is controlled by choice of core, repeating unit, number of generations and terminal functional group. See: U.S. Pat. Nos. 5,527,524; 5,338,532; 4,694,064; 4,568,737; 4,507,466; and PCT patent applications; WO8801179; WO8801178; WO9524221; and WO9502397. xe2x80x9cSTARBURSTxe2x80x9d (Trademark, Dendritech, Inc.) or dense star polyamidoamine dendrimers have been reported to mediate efficient transfection of DNA into mammalian cells (J. F. Kukowska-Latolla et al. (1996) Proc. Natl. Acad. Sci. USA 93:4897-4902 and A. Bielinska et al. (1996) Nucleic Acids Res. 24(11):2176-2182). xe2x80x9cSUPERFECTxe2x80x9d (Trademark, Qiagen, Inc.) or activated dendrimers have been reported to mediate efficient transfection of DNA into mammalian cells (J. Haensler and R. Szoka (1993) Bioconjugate Chem. 4:372-379 and M. X. Tang et al., (1996) Bioconjugate Chem. 7P703-714). PCT patent application WO9524221 relates to bioactive or targeted dendrimer conjugates. PCT patent applications WO9319768 and WO9502397 relate to polynucleotide delivery systems comprising dendrimers.
Transfection agents, including cationic lipids and dendrimers, are not universally effective for transfection of all cell types. Effectiveness of transfection of different cells depends on the particular transfection agent composition and the type of lipid aggregate or dendrimer-complex formed. In general, polycationic lipids are more efficient than monocationic lipids in transfecting eukaryotic cells. Behr, J-P. et al. (1989) Proc. Natl. Acad. Sci. 86:6982-6986, Hawley-Nelson, P., et al. (1993) FOCUS 15:73 and U.S. Pat. No. 5,334,761 (Gebeyehu et al.). Behr et al. and EPO published application 304 111 (1990), for example, describe improved transfection using carboxyspermine-containing cationic lipids including 5-carboxyspermylglycine dioctadecyl-amide (DOGS) and dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide (DPPES). The polycationic liposomal transfection reagents 1,3 dioleoyloxy-2-(6-carboxyspermyl)-propyl-amid (DOSPER, Boehringer-Mannheim) and xe2x80x9cMULTIFECTORxe2x80x9d (Trademark, VennNova, Inc.) are other examples. For transfection, the optimal charge ratio of DNA/dendrimer was found to be between 1:5 and 1:50 and G5 (generation 5)-G10 (generation 10) dendrimers were reported capable of mediating transfection. Transfection efficiency of a given dendrimer varied with cell type, as has been observed with cationic lipid-mediated transfection (J. F. Kukowska-Latolla et al. (1996) Proc. Natl. Acad. Sci. USA 93:4897-4902).
Many biological materials are taken up by cells via receptor-mediated endocytosis. See: Pastan and Willingham (1981) Science 214:504-509. This mechanism involves binding of a ligand to a cell-surface receptor, clustering of ligand-bound receptors, and formation of coated pits followed by internalization of the ligands into endosomes. Both enveloped viruses, like influenza virus and alphaviruses, and non-enveloped viruses, like Adenovirus, infect cells via endocytotic mechanisms. See: Pastan, I. et al. (1986) in Virus Attachment and Entry into Cells, (Crowell, R. L. and Lonberg-Holm, K., eds.) Am. Soc. Microbiology, Washington, p. 141-146; Kielian, M. and Helenius, A. (1986) xe2x80x9cEntry of Alphavirusesxe2x80x9d in The Togaviridae and Flaviviridae, (Schlesinger, S. and Schlesinger, M. J., eds.) Plenum Press, New York p.91-119; FitzGerald, D. J. P. et al. (1983) Cell 32:607-617. Enhancement of dendrimer-mediated transfection of some cells by chloroquine (a lysosomotropic agent) suggests that endocytosis is involved in at least some dendrimer-mediated transfections.
Despite their relative effectiveness, however, successful transfection of eukaryotic cell cultures using polycationic lipid reagents often requires high dosages of nucleic acid (approximately 105 DNA molecules per cell). The introduction of foreign DNA sequences into eukaryotic cells mediated by viral infection is generally orders of magnitude more efficient than transfection with cationic lipid or dendrimer transfection agents. Viral infection of all the cells in a culture requires fewer than 10 virus particles per cell. Although the detailed mechanism of fusion is not fully understood and varies among viruses, viral fusion typically involves specific fusagenic agents, such as viral proteins, viral spike glycoproteins and peptides of viral spike glycoproteins. Vesicular stomatitis virus (VSV) fusion, for example, is thought to involve interaction between the VSV glycoprotein (G protein) and membrane lipids (Schlegel, R. et al. (1983) Cell 32:639-646). The VSV G protein reportedly binds preferentially to saturable receptors such as acidic phospholipid phosphatidylserine (Schlegel, R. and M. Wade (1985) J. Virol. 53(1):319-323). Fusion of influenza virus involves hemagglutinin HA-2 N-terminal fusagenic peptides. See Kamata, H. et al. (1994) Nucl. Acids Res. 22(3):536-537.
Cell binding and internalization can also be enhanced, accelerated or made selective with peptides that bind cell receptors. For example, the penton-base protein of the Adenovirus coat contains the peptide motif RGD (Arg-Gly-Asp) which mediates virus binding to integrins and viral internalization via receptor-mediated endocytosis (Wickham, T. J. et al. (1995) Gene Therapy 2:750-756).
The efficiency of cationic lipid transfections has recently been shown to be enhanced by the addition of whole virus particles to the transfection mixture. See Yoshimura et al. (1993) J. Biol. Chem. 268:2300. Certain viral components may also enhance the efficiency of cationic lipid-mediated transfection. See: U.S. patent applications Ser. Nos. 08/090,290, filed Jul. 12, 1993; and 08/274,397, filed Jul. 12, 1994, now U.S. Pat. No. 5,578,475; incorporated by reference in their entirety herein. The use of peptides from viral proteins to enhance lipid-mediated transfections was also recently suggested by Kamata et al. (1994) Nucl. Acids Res. 22:536. Kamata et al. suggest that xe2x80x9cLIPOFECTINxe2x80x9d-mediated transfections may be enhanced 3-4-fold by adding influenza virus hemagglutinin peptides to the transfection mixture. Despite these positive early indications, results vary as to the effectiveness of including fusagenic or nuclear localization peptides in lipidic transfection compositions. Remy et al. (1995) Proc. Natl. Acad. Sci. USA 92:1744 report that xe2x80x9c[a]ddition of lipids bearing a fusagenic or a nuclear localization peptide head group to the (polycationic lipid-DNA complex) particles does not significantly improve an already efficient system.xe2x80x9d
The present invention is based on the discovery that peptide sequences from viral, bacterial or animal proteins and other sources, including peptides, proteins or fragments or portions thereof can significantly enhance the efficiency of transfection of eukaryotic cells mediated by transfection agents, including cationic lipids and dendrimers. The compositions and methods of the invention comprise peptides, proteins and fragments thereof, modified peptides, modified proteins and modified fragments thereof, peptide conjugates, protein conjugates and conjugates of fragments thereof, including those of fusagenic, membrane-permeabilizing, receptor-ligand, and/or nuclear-localization peptides or proteins, or peptides or proteins that localize to other sub-cellular locations (e.g., mitochondrial localization peptides or proteins), which significantly improve the efficiency of transfection when bound to nucleic acid. In preferred embodiments, peptides, proteins, fragment thereof, or modified peptides, proteins and fragments thereof are bound or added to nucleic acid prior to adding the transfection reagent, although such peptides, proteins, fragments and modifications thereof may be added or complexed with the transfection reagent prior to addition of the nucleic acid. Alternatively, the nucleic acid is combined with the transfection agent prior to addition of the peptide, protein, fragments and modifications thereof. These fusagenic, receptor-ligand, nuclear localization, transport or trafficking, or other peptides can form a noncovalent association or complex with the nucleic acid that is to be introduced into a cell. Complex formation can be enhanced by covalent coupling of the peptide or protein to a DNA-binding group, which can bind to the nucleic acid through conformational or charge interactions and facilitate binding of the peptide to DNA. More generally, nucleic acid-peptide or protein complex formation can be enhanced by covalent coupling of the peptide or protein to a nucleic acid-binding group. Nucleic acids (DNA and RNA and variants thereof) are more efficiently transported into the cell by the transfection agent when bound to peptides or proteins of this invention and can with appropriate choice of peptide or protein be directed to the cell nucleus or to other sub-cellular locations, thus requiring less nucleic acid starting material.
This invention also relates to the covalent coupling of peptides or proteins to the transfection agent, e.g., directly or via an appropriate linking or spacer group to a lipid of the cationic lipid transfection composition (a cationic or neutral lipid) or directly or via an appropriate linking or spacer group to a dendrimer. Of particular interest are conjugated lipids and dendrimers that are covalently linked to fusagenic peptides or proteins, transport or trafficking peptides or proteins, membrane-permeabilizing peptides or proteins and receptor-ligand peptides or proteins. A variety of spacer groups may be used dependent upon the transfection agent and the peptide or protein. For example, spacers may be alkyl, ether, thioether, ester or amide groups.
The cationic lipid compositions of the present invention and the dendrimer compositions of this invention provide significant advantages over prior art compositions, including enhanced transformation frequency.
The present invention provides compositions and methods for transfecting eukaryotic cells, particularly higher eukaryotic cells, with nucleic acids. Nucleic acids, both DNA and RNA, are introduced into cells such that they retain their biological function. Compositions for transfecting eukaryotic cells comprising a peptide-nucleic acid complex or protein-nucleic acid complex and a transfection agent are provided. Transfection compositions of this invention include those in which the transfection agent is any lipid, preferably a cationic lipid, a mixture of cationic lipids or a mixture of cationic lipids and neutral lipids. Transfection compositions of this invention also include those in which the transfection agent is a dendrimer or mixture of dendrimers, as well as mixtures of dendrimers and neutral or cationic lipids. Transfection compositions comprise a peptide or modified-peptide, e.g., a peptide-conjugate, or protein or fragment or portion thereof, modified or conjugated, which may bind nucleic acid and which are fusagenic, membrane-permeabilizing, or which function for nuclear localization, function for transport or trafficking, function for localization to another sub-cellular location, and/or function as a receptor-ligand. Receptor-ligand peptides or proteins of this invention include those that bind to cell surface receptors, membrane receptors or cytosolic receptors and that can function for cell targeting or cell adhesion, and include those that trigger internalization or endocytosis. The peptide- or protein-nucleic acid complex is formed by interacting a peptide or protein or modified peptide or modified protein with nucleic acid or by interacting the peptide or protein with a nucleic acid-transfection agent complex. Modified peptides or proteins include peptides or proteins covalently conjugated to nucleic acid-binding groups. Peptide- or protein-conjugates of this invention also include peptide- or protein-lipid (neutral or cationic) and peptide- or protein-dendrimer conjugates in which the peptide or protein is covalently linked to the transfection agent or a component of the transfection agent.
For non-covalent peptide- or protein-enhanced lipid transfection, the peptide- or protein-nucleic acid complex is subsequently combined with a lipid, preferably a cationic lipid (or a mixture of a cationic lipid and neutral lipid) to form a peptide- or protein-nucleic acid-lipid aggregate which facilitates introduction of the anionic nucleic acid through cell membranes, including the nuclear membrane, or targets the nucleic acid to a particular cell or sub-cellular location. Transfection compositions of this invention comprising peptide- or protein-nucleic acid complexes and lipid can further include other non-peptide agents that are known to further enhance transfection.
For lipid transfection employing a covalent peptide-or protein-lipid conjugate, the peptide- or protein-lipid conjugate is combined with nucleic acid, as is conventional for cationic lipid transfection. The peptide- or protein-lipid conjugate may be first combined in a mixture of non-conjugated cationic and/or neutral lipids and then combined with nucleic acid to form a peptide-or protein-lipid-nucleic acid lipid aggregate which facilitates introduction of the anionic nucleic acid through cell membranes, including the nuclear membrane, or targets the nucleic acid to a particular cell or to a sub-cellular location. Transfection compositions of this invention comprising peptide- or protein-lipid conjugates and nucleic acids can further include other non-peptide or non-protein agents that are known to further enhance transfection.
In an alternative transfection method of this invention employing fusagenic peptides or proteins covalently conjugated to lipids, the peptide- or protein-lipid conjugate is complexed with non-conjugated cationic lipids (or a mixture of cationic and neutral lipids). A sub-cellular localization peptide or protein, preferably a nuclear localization peptide or protein, is complexed to the nucleic acid and the nucleic acid-peptide or protein complex is admixed with the cationic lipid-containing complex comprising covalently conjugated fusagenic peptides or proteins. The resulting mixture exhibits enhanced transfection efficiency.
For dendrimer transfection, the covalent peptide- or protein-dendrimer conjugate is subsequently combined with nucleic acid, as is known in the art for dendrimer-mediated transfection, to form a peptide- or protein-dendrimer-nucleic acid aggregate that facilitates introduction of the anionic nucleic acid through cell membranes, including the nuclear membrane, or targets the nucleic acid to a particular cell or sub-cellular location. When a peptide- or protein-dendrimer conjugate is employed, the peptide or protein is believed, for the most part, to be concentrated at the outer surface of the dendrimer aggregate formed. Transfection compositions of this invention comprising peptide- or protein-dendrimer conjugates and nucleic acid can further include other non-peptide agents that are known to further enhance dendrimer transfection, for example dendrimer transfection can be enhanced by addition of DEAE-dextran and/or chloroquin.
In alternative transfection compositions of this invention employing fusagenic peptides or proteins conjugated to dendrimers, the peptide- or protein-dendrimer conjugate is admixed with a nucleic acid that is itself complexed to a sub-cellular localization peptide or protein, preferably a nuclear localization peptide or protein. The new complex (e.g., Sp-NLS-nucleic acid complexed to VSVG or RGD or E5-dendrimer) is optionally admixed with non-conjugated dendrimers or optionally admixed with a cationic lipid-containing composition. The resulting mixture exhibits enhanced transfection efficiency.
Peptides useful in transfection compositions include, but are not limited to, functional portions of proteins and or polypeptides that are fusagenic, function for nuclear or other sub-cellular localization, function for transport or trafficking, are receptor ligands, comprise cell-adhesive signals, cell-targeting signals, cell-internalization signals or endocytosis signals as well as peptides or functional portions thereof of viral fusagenic proteins, of viral nuclear localization signals, of receptor-ligands, of cell adhesion signals, of cell-targeting signals or of internalization- or endocytosis-triggering signals. Peptides useful in this invention include naturally-occurring peptides, peptides derived from synthetic or engineered proteins or polypeptides, and synthetic analogs or functional equivalents of naturally-occurring peptides. Peptides of this invention include those comprised of the twenty commonly occurring amino acids, as well as rare amino acids, such as homocysteine and ornithine, or D-amino acids or amino acid analogs. Peptides and proteins or this invention can include polyamines such as carboxy spermine. Transfection compositions comprising viral peptides or functional portions of viral peptides of influenza virus, vesicular stomatitis virus, adenovirus and simian virus 40 are of particular interest. Transfecting compositions containing viral peptides (as well as proteins and polypeptides) modified so that they are covalently conjugated to DNA-binding groups, for example, spermine or related polyamines, are also useful in the methods of this invention.
Any proteins (or fragments or portions thereof) may be used in accordance with this invention, either singly or in combination with other proteins or peptides. In a preferred aspect, two or more, three or more, four or more, five or more, six or more, etc. proteins and/or peptides are used in the invention. Additionally, such single or multiple proteins and/or peptides may be used in combination with one or more, two or more, three or more, four or more, five or more, six or more, etc. transfection agents. In another preferred aspect, at least two peptides and/or proteins are used in combination with a transfection agent, preferably at least two transfection agents such as lipids and/or dendrimers.
Proteins useful in transfection compositions include, but are not limited to, receptor ligands, membrane binding and fusion proteins, transport or trafficking proteins, nuclear localizing proteins, nuclear proteins, including proteins derived from chromatin, bacterial internalization-mediating proteins, bacterial toxins or portions of toxins (with toxin portion inactivated), which enter cells and localize to subcellular compartments, membrane-disturbing proteins and antimicrobial proteins. Proteins include those derived from viral, bacterial, animal and other sources. Receptor-ligand proteins which are useful include, but are not limited to, insulin, transferrin, epidermal growth factor, fibroblast growth factor, lactoferrin, and fibronectin. Useful viral membrane binding and fusion proteins include, but are not limited to, the adenoviral proteins penton base, knob, and hexon, the vesicular stomatitis virus glycoprotein (VSVG), the coat proteins from semliki forest virus and the influenza hemagglutinin (HA). Viral transport or trafficking proteins include, but are not limited to, HIV Tat, hepatitis B virus core protein, and herpes simplex virus VP22. A list of nuclear and chromatin proteins which are useful includes, but is not limited to, the histone proteins, especially H1 and H2, the xe2x80x9chigh mobility groupxe2x80x9d proteins, especially HMG 1 and 17, protamine and hn RNP A1. Bacterial internalization proteins include, but are not limited to, invasin and internalin and proteins with similar functions derived from Listeria and Myobacterium tuberculosis. Bacterial toxins which enter cells and localize to subcellular compartments include, but are not limited to, Pseudomonas endotoxin A, Diphtheria toxin, and Shigella toxin. In each case, the bacterial toxin function of these proteins and polypeptides is inactivated to avoid detriment to transfected cells. Membrane-disturbing and anti-microbial proteins (some derived from venoms) include, but are not limited to, melittin, magainin, gramicidin, cecropin, defensins, protegrins, tachyplesins, thionins, indolicidin, bactenecin, drosomycin, apidaecins, cathelicidin, bacteriacidal/permeability-increasing protein (BPI), nisin, and buforin.
Inclusion of a peptide- or protein-nucleic acid complex or a modified peptide- or protein-nucleic acid complex in a cationic lipid transfection composition can significantly enhance transfection (by 2-fold or more) of the nucleic acid compared to transfection of the nucleic acid mediated by the cationic lipid alone. Enhancement of dendrimer transfection by peptides or proteins or modified peptides or modified proteins or fragments thereof is pronounced in a wide variety of cell lines, including human primary cell lines and in cell lines that are generally considered by those in the art to be xe2x80x9chard-to-transfect.xe2x80x9d
Monovalent or polyvalent cationic lipids are employed in cationic lipid transfecting compositions. Preferred monovalent cationic lipids are DOTMA (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl ammonium chloride), DOTAP (1,2-bis(oleoyloxy)-3,3-(trimethylammonium)propane), DMRIE (1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide) or DDAB (dimethyl dioctadecyl ammonium bromide). Preferred polyvalent cationic lipids are lipospermines, specifically DOSPA (2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoro-acetate) and DOSPER (1,3-dioleoyloxy-2-(6carboxy spermyl)-propyl-amid, and the di- and tetra-alkyl-tetra-methyl spermines, including but not limited to TMTPS (tetramethyltetrapalmitoyl spermine), TMTOS (tetramethyltetraoleyl spermine), TMTLS (tetramethlytetralauryl spermine), TMTMS (tetramethyltetramyristyl spermine) and TMDOS (tetramethyldioleyl spermine). Cationic lipids are optionally combined with non-cationic lipids, particularly neutral lipids, for example lipids such as DOPE (dioleoylphosphatidylethanolamine), DPhPE (diphytanoylphosphatidylethanolamine) or cholesterol. A cationic lipid composition composed of a 3:1 (w/w) mixture of DOSPA and DOPE or a 1:1 (w/w) mixture of DOTMA and DOPE are generally useful in transfecting compositions of this invention. Preferred transfection compositions are those which induce substantial transfection of a higher eukaryotic cell line.
Inclusion of a peptide- or protein-nucleic acid or modified peptide- or protein-nucleic acid complex in a dendrimer transfection composition can significantly enhance transfection (by 2-fold or more) of the nucleic acid compared to transfection of the nucleic acid mediated by the dendrimer alone or in combination with DEAE-dextran or chloroquine or both. Enhancement of transfection by peptides, proteins, modified peptides or modified proteins is pronounced in a wide variety of cell lines, including human primary cell lines and in cell lines that are generally considered by those in the art to be xe2x80x9chard-to-transfect.xe2x80x9d
In general, any dendrimer that can be employed to introduce nucleic acid into any cell, particularly into a eukaryotic cell, is useful in the improved transfection compositions and methods of this invention. Dendrimers of generation 5 or higher (G5 or higher) are preferred, with those of generation between G5-G10 being of particular interest. Dendrimers of this invention include those with NH3 or ethylenediamine cores, GX(NH3) or GX(EDA), where X=the generation number. Dendrimers where X=5-10 being preferred. Dendrimers of this invention include those in which the repeating unit of the internal layers is a amidoamine (to form polyamidoamines, i.e. PAMAMs). Dendrimers of this invention include those in which the terminal functional groups at the outer surface of the dendrimer provides a positive charge density, e.g., as with terminal amine functional groups. The surface charge and the chemical nature of the outer dendrimer surface can be varied by changing the functional groups on the surface, for example, by reaction of some or all of the surface amine groups. Of particular interest are dendrimers that are functionalized by reaction with cationic amino acids, such as lysine or arginine. Grafted dendrimers as described, for example in PCT applications WO 9622321 and WO9631549 and noted in U.S. Pat. No. 5,266,106, can be employed in the compositions and methods of this invention. Activated dendrimers (J. Haensler and R. Szoka (1993) Bioconjugate Chem. 4:372-379 and M. X. Tang et al., (1996) Bioconjugate Chem. 7P703-714) can also be employed in the composition and methods of this invention.
The methods of the present invention involve contacting any cell, preferably a eukaryotic cell, with a transfection composition comprising a peptide, a protein or fragment or portion thereof, including a fusagenic, membrane-permeabilizing, transport or trafficking sub-cellular-localization, or receptor-ligand peptide or protein, optionally conjugated to a nucleic acid-binding group, or optionally conjugated to the transfection agent (lipid or dendrimer) wherein said peptide or protein or modified peptide or protein is non-covalently associated with the nucleic acid. In one embodiment, a peptide- or protein-nucleic acid complex (where the peptide or protein can be conjugated to a nucleic-acid binding group) is formed and then combined with a cationic lipid for transfection. In a related embodiment, a peptide- or protein-lipid conjugate is combined optionally with other lipids, including any appropriate cationic lipid, and then combined with nucleic acid for transfection. In another related embodiment, a nucleic acid-lipid complex is formed and then combined with a peptide or protein for transfection. In a second embodiment, a peptide- or protein-nucleic acid complex (where the peptide or protein can be conjugated to a nucleic-acid binding group) is formed and then combined with a dendrimer for transfection. In a related embodiment, a peptide-dendrimer conjugate is combined optionally with other dendrimers and then combined with nucleic acid for transfection. In another related embodiment, a nucleic acid-dendrimer complex is formed and then combined with a peptide or protein for transfection. Dendrimers and/or peptide-conjugated dendrimers can be combined with cationic lipids and cationic lipid composition to obtain improved nucleic acid transfection compositions. In accordance with the invention, multiple peptides and/or proteins may be added to accomplish transfection.
Methods of this invention employ among others, viral peptides or proteins of influenza virus, adenovirus, Semliki forest virus, HIV, hepatitis, herpes simplex virus, vesicular stomatitis virus or simian virus 40 and more specifically an RGD-peptide sequence, an NLS peptide sequence and/or a VSVG-peptide sequence and to modified peptides or proteins of each of the foregoing. Methods of this invention are applicable to transfection of adherent or suspension cell lines, in general to animal cell lines, specifically to mammalian, avian, reptilian, amphibian and insect cell lines and more specifically to animal primary cell lines, human primary cell lines, stem cell lines, and fibroblasts, as well as to cells in vivo in living organisms.
In one specific embodiment, a transfection-enhancing peptide or protein is first bound to a nucleic acid to be introduced into a cell. The peptide- or protein-nucleic acid complexes are then admixed with a transfection agent (or mixture thereof) and the resulting mixture is employed to transfect cells. Preferred transfection agents are cationic lipid compositions, particularly monovalent and polyvalent cationic lipid compositions, more particularly xe2x80x9cLIPOFECTIN,xe2x80x9d xe2x80x9cLIPOFECTACE,xe2x80x9d xe2x80x9cLIPOFECTAMINE,xe2x80x9d xe2x80x9cCELLFECTIN,xe2x80x9d DMRIE-C, DMRIE, DOTAP, DOSPA, and DOSPER, and dendrimer compositions, particularly G5-G10 dendrimers, including dense star dendrimers, PAMAM dendrimers, grafted dendrimers, and dendrimers known as dendrigrafts and xe2x80x9cSUPERFECT.xe2x80x9d
In a second specific transfection method, a transfection-enhancing peptide or protein is conjugated to a nucleic acid-binding group, for example a polyamine and more particularly a spermine, to produce a modified peptide or protein which is then bound to the nucleic acid to be introduced into the cell. The modified peptide-nucleic acid complex is then admixed with a transfection agent (or mixture thereof) and the resulting mixture is employed to transfect cells. In particular, the peptide or protein is covalently conjugated to a spermine, the spermine-modified peptide or protein is complexed with nucleic acid and admixed with a cationic lipid. Preferred transfection agents are cationic lipid compositions, particularly monovalent and polyvalent cationic lipid compositions, more particularly xe2x80x9cLIPOFECTIN,xe2x80x9d xe2x80x9cLIPOFECTACE,xe2x80x9d xe2x80x9cLIPOFECTAMINE,xe2x80x9d xe2x80x9cCELLFECTIN,xe2x80x9d DMRIE-C, DMRIE, DOTAP, DOSPA, and DOSPER, and dendrimer compositions, particularly G5-G10 dendrimers, including dense star dendrimers, PAMAM dendrimers, grafted dendrimers, and including dendrimers known as dendrigrafts.
In a third specific embodiment, a mixture of one or more transfection-enhancing peptides, proteins, or protein fragments, including fusagenic peptides or proteins, transport or trafficking peptides or proteins, receptor-ligand peptides or proteins, or nuclear localization peptides or proteins and/or their modified analogs (e.g., spermine modified peptides or proteins) or combinations thereof are mixed with and complexed with nucleic acid to be introduced into a cell. The peptide-nucleic acid complexes are then admixed with transfection agent and the resulting mixture is employed to transfect cells.
In another specific embodiment, a component of a transfection agent (lipids, cationic lipids or dendrimers) are covalently conjugated to selected peptides, proteins, or protein fragments directly or via a linking or spacer group. Of particular interest in this embodiment are peptides or proteins that are fusagenic, membrane-permeabilizing, transport or trafficking, or which function for cell-targeting. The peptide- or protein-transfection agent complex is combined with nucleic acid and employed for transfection.
The transfection compositions and methods of the present invention can be applied to in vitro and in vivo transfection of cells, particularly of eukaryotic cells, and more particularly to transfection of higher eukaryotic cells, including animal cells. The methods of this invention can be used to generate transfected cells which express useful gene products. The methods of this invention can also be employed as a step in the production of transgenic animals. The methods of this invention are useful as a step in any therapeutic method requiring introduction of nucleic acids into cells including methods of gene therapy and viral inhibition and for introduction of antisense or antigene nucleic acids or ribozymes or RNA regulatory sequences or related inhibitory or regulatory nucleic acids into cells. In particular, these methods are useful in cancer treatment, in in vivo and ex vivo gene therapy, and in diagnostic methods.
The transfection compositions and methods of this invention comprising peptides, proteins, peptide or protein fragments or modified peptides or modified proteins, can also be employed as research reagents in any transfection of eukaryotic cells done for research purposes. The transfection compositions can, with appropriate choice of physiologic medium, be employed in therapeutic and diagnostic applications.
Transfection agents and transfection-enhancing agents of this invention can be provided in a variety of pharmaceutical compositions and dosage forms for therapeutic applications. For example, injectable formulations, intranasal formulations and formulations for intravenous and/or intralesional administration containing these complexes can be used therapy.
In general the pharmaceutical compositions of this invention should contain sufficient transfection agent and any enhancing agents (peptide, protein, etc.) to provide for introduction of a sufficiently high enough level of nucleic acid into the target cell or target tissue such that the nucleic acid has the desired therapeutic effect therein. The level of nucleic acid in the target cell or tissue that will be therapeutically effective will depend on the efficiency of inhibition or other biological function and on the number of sites the nucleic acid must affect.
The dosage of transfection agent administered to a patient will depend on a number of other factors including the method and site of administration, patient age, weight and condition. Those of ordinary skill in the art can readily adjust dosages for a given type of administration, a given patient and for a given therapeutic application.
It will be appreciated by those of ordinary skill in the art that the transfection composition should contain minimal amounts of inhibitory components, such as serum or high salt levels, which may inhibit introduction of nucleic acid into the cell, or otherwise interfere with transfection or nucleic acid complexation. It will also be appreciated that any pharmaceutical or therapeutic compositions, dependent upon the particular application, should contain minimal amounts of components that might cause detrimental side-effects in a patient.
Components of the transfection compositions of this invention can be provided in a reagent kit. In general, the kit comprises a transfection agent and a transfection-enhancing peptide, protein or fragment thereof. In one embodiment, a kit comprises individual portions of cationic lipid and peptide, protein or fragment thereof or modified peptide, protein or fragment thereof. In a second embodiment, a kit comprises individual portions of dendrimer and peptide, protein or fragments thereof or modified peptide, protein or fragments thereof. Cationic lipid transfection kits can optionally include neutral lipid as well as other transfection-enhancing agents or other additives, and the relative amounts of components in the kit may be adjusted to facilitate preparation of transfection compositions. Kit components can include appropriate medium or solvents for other kit components. Cationic lipid transfection kits comprising a monocationic or polycationic lipid composition including a neutral lipid and a modified peptide or protein are preferred. Dendrimer transfection kits can optionally include other transfection enhancing agents, such as DEAE-dextran and/or chloroquine, as well as other additives and the relative amounts of components in the kit may be adjusted to facilitate preparation of transfection compositions. Dendrimer transfection kits comprising a G5-G10 dendrimer or a Lys- or Arg-modified dendrimer or dendrigraft or an activated dendrimer in combination with a peptide or protein or a modified peptide or protein are preferred. Kits provided by this invention include those comprising an individual portion of a polycationic lipid composition comprising DOSPA and DOPE or a monocationic lipid composition comprising DOTMA and DOPE and a portion of modified peptide, particularly a spermine-modified peptide. Kits provided by this invention include those comprising an individual portion of a dendrimer and a portion of a spermine-modified peptide.
In related embodiments, kits of this invention can comprise a peptide- or protein-lipid conjugate or a peptide- or protein-dendrimer conjugate in combination with non-conjugated lipids, non-conjugated dendrimers and other agents to facilitate transfection.
Kits of this invention can include those useful in diagnostic methods, e.g., diagnostic kits which in addition to transfection agent and transfection-enhancing agents (e.g., proteins, peptides, and fragments and modifications of peptides and proteins) can contain a diagnostic nucleic acid. A diagnostic nucleic acid is a general term for any nucleic acid which can be employed to detect the presence of another substance (most generally an analyte) in a cell. For example, when transfected into a cell a diagnostic nucleic acid may increase or decrease expression of a gene therein in response to the presence of another substance in the cell (e.g., a protein, small molecule, steroid, hormone, or another nucleic acid). Diagnostic nucleic acids also include those nucleic acids that carry some label or otherwise detectable marker to a particular target cell or target tissue for detection of the target cell or tissue or for detection of a substance in the target cell or tissue.
Nucleic acids that can be transfected by the methods of this invention include DNA and RNA of any size from any source comprising natural bases or non-natural bases, and include those encoding and capable of expressing therapeutic or otherwise useful proteins in cells, those which inhibit undesired expression of nucleic acids in cells, those which inhibit undesired enzymatic activity or activate desired enzymes, those which catalyze reactions (ribozymes), and those which function in diagnostic assays (e.g., diagnostic nucleic acids). Therapeutic nucleic acids include those nucleic acids that encode or can express therapeutically useful proteins, peptides or polypeptides in cells, those which inhibit undesired expression of nucleic acids in cells, those which inhibit undesired enzymatic activity or activate desired enzymes in cells.
The compositions and methods provided herein can also be readily adapted in view of the disclosure herein to introduce biologically-active macromolecules other than nucleic acids including, among others, polyamines, polyamine acids, polypeptides and proteins into eukaryotic cells. Other materials useful, for example as therapeutic agents, diagnostic materials, research reagents, which can be bound to the peptides and modified peptides and introduced into eukaryotic cells by the methods of this invention.